The emergence and spread of chloroquine resistance (CQR) in Plasmodium falciparum has compounded the global problem of malaria, with significant increases in malaria mortality reported in Africa. Our prior grant addressed the hypothesis that pfcrt, which encodes a digestive vacuole (DV) transmembrane protein, was the primary parasite determinant of CQR. This was confirmed by transaction experiments that exchanged mutant and wild type pfcrt alleles, causing a complete phenotypic change in CQ response. Studies have also revealed extraordinary region-specific allelic diversity, implicated pfcrt alleles in resistance to multiple antimalarials, and suggested that mutant pfcrt gametocytes are preferentially transmitted to the mosquito host. Here, we will test whether mutant pfcrt alleles can confer resistance to multiple drugs, define their impact on parasite transmission, and leverage our isogenic pfcrt-modified lines to probe the CQR mechanism and identify functional properties that distinguish mutant and wild type pfcrt. Specific Aim 1 will test the hypothesis that the global diversity of pfcrt alleles reflects their key role in mediating resistance to CQ and other drugs used regionally to treat CQ-resistant malaria. This will be achieved using allelic exchange techniques to engineer parasites expressing geographically diverse pfcrt alleles with selected mutations, followed by phenotypic characterization of isogenic lines. These experiments benefit from our development of a highly improved transfection system that uses mycobacteriophage integrase to mediate rapid site-specific allelic exchange in P. falciparum. Specific Aim 2 will test the hypothesis that the enhanced gametocyte production and transmissibility frequently observed with CQ-resistant infections is imparted by mutant pfcrt. Isogenic, pfcrt-modified CQ-resistant and CQ- sensitive clones will be quantitatively assayed for gametocyte development, gamete exflagellation, transmission and oocyst production in infected mosquitoes in the presence or absence of CQ. Specific Aim 3 will implement biochemical assays to test the hypothesis that CQR is primarily attributable to energy- dependent active CQ efflux that is conferred by PfCRT mutations. These studies will also assess models of passive drug leak and altered DV pH. Using isogenic pfcrt -modified and control lines harboring distinct CQ phenotypes, we propose experiments that will clarify key mechanistic features of pfcrf-mediated CQR and relate this to functional differences between pfcrt alleles and individual polymorphisms. These studies are designed to elucidate the contribution of mutant pfcrt to multidrug resistance, generate novel insights into the transmission and spread of resistance, and identify key functional properties of mutant pfcrt alleles and the CQR mechanism. The results should be of significant benefit to public health programs aimed at identifying and combating drug-resistant malaria.